摘要
Hedgehog信号转导通路下游转录因子Gli1在人神经胶质瘤中对水通道蛋白AQP1调控作用的研究
研究背景和目的
Hedgehog(Hh)信号转导通路是在脊椎动物的中枢神经系统发育中起至关重要作用的一条信号通路,其在胚胎早期可以通过通路各组分不同时间、空间的特异性表达对多种细胞的命运决定产生影响,并且可以与Wnt、FGFs、BMPs等信号通路产生相互作用来实现不同细胞定向分化的精细调节。Hedgehog信号的调节紊乱在胚胎期会造成神经系统的发育障碍和畸形,在已经成熟的个体则会导致肿瘤的发生。Gli1(Glioma-associated Oncogene Homolog 1,Gli1)最初是在研究神经胶质瘤时发现并命名的一个基因,其编码的Gli1蛋白是Hh信号途径最重要的下游转录因子之一,也是Hh信号最终的响应者和功能的执行者。Hh-Gli1信号通路在神经胶质瘤中处于一种调节紊乱的异常激活状态,在胶质瘤的发生发展中起着重要作用,其上游配体Shh信号的存在对于胶质瘤的生长及肿瘤干细胞的自我更新能力的维持有着重要作用,其下游转录因子Gli1可以通过调节
CyclinD1,Bcl-2的表达水平参与胶质瘤细胞的增殖和凋亡,同时也与胶质瘤的恶性程度和恶性生物学行为的维持密切相关。
Aquaporins(水通道蛋白,AQPs)是一类分布在细胞膜表面,能够特异的对水分子进行快速高效跨膜转运的小分子通道蛋白家族。在中枢神经系统中主要分布的水通道蛋白是AQP1。近年来的研究发现AQPs与神经系统的一些疾病尤其是肿瘤性疾病的进展有着密切的关系,AQP1在胶质瘤细胞向周围组织的侵袭生长及瘤周组织的脑水肿形成方面都发挥了重要的作用,虽然已经有大量的研究阐明了AQP1是如何通过自身的通道结构对水分子进行跨膜转运的,但是对其本身表达水平的高低受何种机制的调控尤其是它与主要信号通路之间的关系仍然知之甚少。
本研究拟从一种中枢神经系统最常见的肿瘤——神经胶质瘤入手,研究Hedgehog通路的异常激活与水通道蛋白AQP1之间的关系,旨在证明Hedgehog通路下游转录因子Gli1对AQP1的表达具有调控作用,阐明这种调控是在何种水平、以何种方式进行,并对这种调控作用对神经胶质瘤细胞恶性生物学行为产生的影响进行了初步探讨。
实验方法
1.用免疫组织化学、免疫荧光共定位实验和Western blot等方法研究神经胶质瘤标本中Hedgehog通路的激活情况以及AQP1的表达情况,并对两者之间的相关程度进行分析;
2.构建包含AQP1启动子区全长的Luciferase报告基因质粒AQP1-Luc-F,将Gli1-myc与报告质粒共转细胞,荧光素酶报告基因系统检测Gli1对AQP1-Luc-F激活程度的影响;
3.将Gli1过表达质粒瞬转细胞,收获不同时间点及不同转入剂量的样品,RT-PCR观察转录水平、Western blot观察蛋白水平AQP1的表达变化;
4.设计合成Gli1基因特异的干扰片段,构建干扰载体,体外验证有效后,研究干扰掉Gli1后对AQP1报告基因活性及胶质瘤细胞系中AQP1表达的影响;
5.建立分别稳定转染空载体、过表达Gli1、及稳定干扰Gli1的三种神经胶质瘤细胞系:U251-PC3.1,U251-Gli1,U251-PG1,Western Blot方法检测三种细胞系中AQP1表达水平的变化;
6.将报告质粒转入三种不同的稳定细胞系中,检测AQP1转录活性的变化;
7.分析AQP1启动子区,根据Gli1的核心结合序列设计引物,运用染色质免疫共沉淀(ChIP)技术研究Gli1与AQP1启动子区的结合情况;
8.根据ChIP实验结果,利用重叠延伸PCR技术构建AQP1启动子区突变报告基因质粒,转入相应的稳定细胞系中,利用报告基因检测确定在Gli1对AQP1调控中起关键作用的启动子区段位置,阐明调控作用的机制;
9.利用Transwell和Wound healing assay实验比较不同细胞系侵袭能力的差异,评价Gli1在胶质瘤细胞系中调控AQP1表达所引起的生物学功能变化。
实验结果
1.神经胶质瘤组织标本中Gli1和AQP1的表达与对照正常组织相比都呈现出异常高表达的状态,并且两者之间的高表达具有特异的相关性;
2.外源瞬时转入及稳定表达Gli1都可以在转录和翻译水平上调AQP1的表达,并能显著增强含有AQP1启动子区全长的报告基因的活性,;
3.特异性干扰Gli1的表达则可以降低AQP1的表达并抑制AQP1的报告基因活性; 4. Gli1可以和AQP1启动子区中的保守调节序列相结合,其中AQP1启动子区-406—-398位的保守结合序列对维持Gli1的调节作用至关重要;
5. Gli1表达水平不同的神经胶质瘤细胞系表现出不同的侵袭转移能力,在侵袭能力较低的细胞系中外源性转入AQP1可以使其侵袭能力得到恢复提高,提示神经胶质瘤中Gli1可能通过上调AQP1的表达来增强肿瘤细胞的恶性生物学特性。
结论
本研究首次发现了神经胶质瘤中Hedgehog通路异常活化与AQP1异常高表达两种现象之间的关系,并且证实Hh通路下游转录因子Gli1可以通过与AQP1启动子区的结合在转录和翻译水平对其表达进行调控,其中AQP1启动子区-406—-398段的DNA序列在这一调控作用中发挥重要作用。在胶质瘤细胞中这一调控作用的生物学意义是:Gli1可以通过上调AQP1来增强胶质瘤细胞的恶性侵袭能力。
基于Jervine结构修饰的靶向Hedgehog通路小分子抑制剂的筛选研究
研究背景和目的
Hedgehog信号转导通路(Hh通路)是一条高度保守的,在生物的胚胎发育及多种疾病的发生发展过程中起重要作用的信号通路。近年来的研究表明,在多种人类恶性肿瘤性疾病中(如脑胶质细胞瘤、基底细胞癌、胰腺癌、非小细胞肺癌、前列腺癌等)该通路都呈现出异常的活化状态,并且对肿瘤的发生及发生后肿瘤组织的生长和恶性生物学特性的维持起着重要作用。人们在研究Hh通路与肿瘤之间关系时发现,特异性抑制Hh通路可以抑制多种肿瘤细胞的增殖以及向其他器管侵袭转移的能力。因此,Hedgehog信号通路被认为是一条比较理想的可以作为肿瘤靶向治疗的信号途径,针对Hh通路的小分子抑制剂的开发及研究工作方兴未艾,逐渐成为国际研究的热点。
Jervine(介芬胺)是从藜芦属植物中提取的一种小分子天然产物,在化学结构上属于一种甾体类生物碱。Jervine可以与Hh通路上游激活因子Smo特异性结合而抑制该通路信号的传递,进而发挥抑制肿瘤细胞的增殖的效应,是一种具有潜在临床应用价值的小分子抑制剂。但是Jervine作为一种天然产物,其抑制Hh通路活性的作用并不是很强且具有较大的化学毒性,这一缺陷限制和延缓了其作为一种新型抗肿瘤药物的研发与应用。
本研究拟以Jervine为先导化合物,通过结构修饰和改造的方法制备系列目标衍生物;然后对制备的衍生物进行抑制Hh通路的特异性筛选及抗肿瘤活性筛选,并与已有的Hedgehog通路抑制剂的活性进行比较,最终期望获得一到两个高效、低毒、高特异性的Hedgehog通路抑制剂,并对结构修饰规律作出总结,为新型抗肿瘤药物的研发提供基础。
实验方法
1.结构修饰衍生物的获得:以Jervine为底物,分别经醚化、N-烃化、N-酰化,N-甲基化、3-OH氯代等特异性化学修饰得到一系列衍生化合物;
2.衍生物高通量筛选平台的构建:在Hh通路组成性激活的NIH/3T3细胞系中稳定转染可以反应Hh通路激活程度的报告基因Hh-reporter,将该细胞系命名为NIH/3T3-R,并对该细胞系反映Hh通路激活程度的敏感性进行鉴定;
3.初次筛选:以步骤2中构建的稳定细胞系为筛选工具,将步骤1中得到的衍生物以8uM浓度作用于体外培养的C3H-3T3-R,36h后测定报告基因数值,观察各化合物对Hh通路的抑制效应,得到可以相对特异性抑制Hh通路活性的化合物;
4.二次筛选:细胞活性检测再次筛选步骤3中得到的化合物对体外培养的NIH/3T3-R的细胞毒性,排除初筛过程中报告基因数值的降低是由于化合物本身的毒性导致细胞死亡所致,得到对细胞增殖抑制不明显,但可以显著抑制报告基因活性的毒性低、特异性好的衍生物;
5. RT-PCR及Western blot检测步骤4中得到的化合物对NIH/3T3细胞中Hh通路下游转录因子Gli1表达的抑制作用,进一步验证化合物的特异性;
6. CCK8法检测经二次筛选得到的衍生物在体外对肿瘤细胞的增殖抑制作用;裸鼠荷瘤实验,将得到的化合物以不同剂量灌胃给药,观察其对体内肿瘤的生长抑制作用;最终得到具有较高的抗肿瘤活性、低毒高效的Hh通路抑制剂;
实验结果
1.以Jervine为先导化合物进行化学修饰和改造,分离纯化后共得到95个衍生物;
2.报告基因法初筛,检测修饰后的化合物对Hh通路的相对抑制作用,与阳性对照药物Cyclopamine(环杷明)相比较,共有20个化合物显示出与其类似或更佳的抑制作用;
3.用CCK8法再次检测初筛得到的化合物对体外培养细胞的增殖活性的影响,剔除细胞毒性较大的衍生物。再次筛选后得到4个毒性较低,特异性较好的化合物,分别为:33号化合物,IC50=1.48uM;35号化合物,IC50=10.2uM;40号化合物,IC50=4.81 uM;95号化合物,IC50=6.5uM;
4. RT-PCR及Western blot结果显示经两次筛选后得到的4个化合物可以有效降低Hh通路下游转录因子Gli1的mRNA及蛋白表达水平,提示其具有良好的特异性抑制Hh通路活性的作用;
5.在体外肿瘤细胞增殖抑制实验中,筛选到的化合物显示出选择性抑制Hh通路依赖的肿瘤细胞的特性;裸鼠荷瘤实验中四种化合物均表现出较好的抑制肿瘤生长的作用,其对体内肿瘤生长的抑制效率如下:33号化合物为48.26%,35号化合物为42.61%,40号化合物为42.17%,95号化合物为40.64%;其中统计学分析结果表明33号化合物对肿瘤生长的抑制作用要优于阳性对照化合物环杷明。
结论
本研究以Jervine为先导化合物,首先经特异性结构修饰改造得到一系列衍生物,然后对衍生物在抑制Hh通路的特异性、体内外毒性、抗肿瘤活性等方面的特性进行连续筛选,最终得到四个特异性较好、毒性较低、抗肿瘤活性较强的阳性化合物,其中有三个化合物在上述特性上与阳性对Cyclopamine(环杷明)类似,一个化合物要优于环杷明。对所得阳性化合物的结构改造位点与先导化合物Jervine的结构进行比较分析可以得出以下规律:3-OH氧化能够增强化合物的特异性及抗肿瘤活性,但不明显;对化合物毒性影响亦不显著;化合物的毒性、针对Hh通路的特异性以及抗肿瘤活性主要与与结构中哌啶环的NH密切相关,对哌啶环的NH进行结构修饰是发现高效低毒衍生物的重要方向。
PartⅠHedgehog-Gli1 Signaling Mediates Regulation of AQP1 Expression in Human Glioma
Glioma, especially Glioblastoma multiformes (GBMs) express increased aquaporin1(AQP1) compared to normal brain. AQPs may contribute to brain edema, cell motility, and tumor angiogenesis. Although the physiology of AQP1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP1 gene expression. Furthermore the relationship between the regulation of the AQP1 expression and the major intracellular signaling pathways is also poorly concerned. Hedgehog signaling pathway plays an important role in the genesis of Glioma and is also required for sustained glioma growth and survival. In the present study, we report that Gli1, the transcriptional activator of the Hedgehog pathway, is coexpressed with AQP1 in human Glioma tissues and can enhance AQP1 gene transcription by binding to conserved core Gli-binding site in the 5’-flanking promoter region of the AQP1. Transfection 293T and U251 with Gli1 can markedly increase the AQP1 promoter reporter activity and the AQP1expression level .However, suppressing the Gli1 expression using the specific RNAi can abort this effect. The ChIP and reporter mutation analysis showed that the -406～-398 region of the AQP1 promoter is essential for regulation activity of Gli1. Then we investigated the migration and invasion ability of cell lines expressing different levels of Gli1 and AQP1. The results demonstrated that restoring the AQP1 expression in the Gli1-knockdown cell line U251-PG1 can abort the decrease in the migration and invasion ability, which suggested that Gli1 may contribute to the invasion ability of glioma cells through AQP1. The upregulation of Gli1 and its consequences of increased AQP1 in gliomas may provide a new therapeutic target, either as a cell surface marker or as a functional intervention.
Screening for the more effective Small-molecule Inhibitors of the Hedgehog signaling Pathway on the basis of Jervine
The Hedgehog signaling pathway plays an important role in the embryonic patterning and development of many tissues and somatic structures as well as maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in several cancers, including medulloblastoma, rhabdomyosarcoma, melanoma, basal cell carcinoma, and breast, lung, liver, stomach, prostate, and pancreatic cancers.Blocking the Hedgehog pathway can prevent tumor cell proliferation and induce apoptosis. Inhibition of the aberrant Hh-Gli pathway has thus emerged as an attractive target for anticancer therapy. Jervine is a natural steroidal alkaloids from plants of the genus Veratrum that block the Hh signaling pathway by antagonizing Smo function. However, as a natural product, Jervine is not as active as expected and the cytotoxicity of it is also a problem. Here we generated a series of Jervine analogues by structure modification and constructed a cell-based reporter assay of Gli mediated transcription to search for products that could more effectively inhibit Hh signaling. Foure more active modified compounds were identified and importantly, the discovered compounds efficiently inhibited in vitro tumor cell proliferation in a Hh-dependent manner and successfully blocked cell growth in an in vivo xenograft model using human prostate cancer cells harboring down stream activation of the Hh pathway.
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